16S/18S/ITS Amplicon Sequencing

Details

FAQ

Case Study

As a leading provider of NGS services and a partner of Illumina, CD Genomics offers a portfolio of solutions for metagenomics sequencing. 16S/18S/ITS amplicon sequencing is characterized by cost-efficiency, high-speed and practicability to help you identify and investigate the microbial community. With over 10 years of experience, we can totally meet your project requirements and budgets in the exploration of microbial biodiversity.

Introduction of 16S/18S/ITS Amplicon Sequencing

16s rRNA gene, a highly conserved component, is the most widely used gene marker for genus and species identification and taxonomic significance in bacteria and archaea. The estimated substitution rate for hypervariable regions is approximately 7000 times higher than the highly conserved ones, which contains abundant taxonomic information based on these genetic differences. Therefore, the 16S gene amplicons obtained from PCR can be used to deduce taxonomic identifications based upon bioinformatics alignments.

Nevertheless, 18S rRNA is commonly used in fungi for phylogenetics since it has more hypervariable domains than 16S. In addition to this, the ITS (internal Transcribed Spacer) region (including 5.8S), removed in the posttranscriptional process of nuclear rRNA cistron, has been widely regarded as a universal fungi barcode marker for a successful identification for the broadest range of fungi. And compared to 18S, ITS is more variable and hence more suitable as the genetic marker for measuring intraspecific genetic diversity.

16S/18S/ITS Amplicon Sequencing has now been a well-established method for microbial identification and phylogeny studies of samples from complicated microbiomes or environments. In addition to next-generation sequencing platforms, CD Genomics also provides full-length 16S/18S/ITS amplicon sequencing by using PacBio SMRT sequencing technology.

Advantages of 16S/18S/ITS Amplicon Sequencing

The most common housekeeping genetic markers with conserved and variable regions.

Our highly experienced expert team and strict quality control following every procedure to ensure the comprehensive and accurate results. And a PCR-free protocol is optional for a fast workflow and superior data quality upon your request. The typical turnaround time for the whole workflow is approximately 30 working days.

Service Specification

Sample requirements and preparation

Samples sources including human, animals, natural environments and industrial environments, as well as DNA and PCR products

Species annotation, species profiling histogram, heat map and phylogenetic tree

Alpha diversity, Beta diversity, Meta-analysis

… (more upon request)

Analysis pipeline

At CD Genomics, with multiple specialists and years of experience in this area, we guarantee you high-quality data and integrated bioinformatics analyses. If you are interested in what CD Genomics can do with the 16S/18S/ITS Amplicon Sequencing, please do not hesitate to contact us. We are more than happy to be of assistance!

1. Which region is optimal for 16S amplicon sequencing?

We provide multiple primers sets for 16S/18S/ITS amplicon sequencing (please refer to Additional Resources for details), which are based on scientific papers. There are no conclusions about which primer set is optimal. According to some papers, the V4 region of 16S is relatively suitable for diversity analysis in microbial communities.

2. What are the advantages of HiSeq platforms compared to MiSeq platforms?

Compared to MiSeq platforms, HiSeq platforms can receive more accurate sequences (HiSeq 2500 system generates greater than 85% of bases above Q30) in a higher throughput manner. And the assembly efficiency in hypervariable regions with high GC content and/or with a high repetition rate is increased by 25%.

3. Are replicated samples necessary? If so, how many are appropriate?

Replicated samples are necessary for a more qualified outcome in view of individual difference, statistical error, and deviated samples. Generally, at least three replicated samples are required, and five are better. As for samples with high specificity, like faecal, more than 10 samples are recommended.

3. Is ITS or 18S amplicon sequencing fitter for the diversity analysis of eukaryotic microbes?

Generally, ITS amplicon sequencing is fitter for the research on fungi diversity with a high accuracy in species annotations, while 18S amplicon sequencing aims at the diversity of eucaryotic microbes, which provides a wider spectrum of species annotations with a relatively decreased accuracy. If you hardly know how to choose, our experienced scientist will work with you closely to provide assistance in project design.

4. What are the differences between amplicon sequencing and metagenomics sequencing?

Amplicon sequencing employs versatile markers, such as 16S, 18S, and ITS, to provide a cost-effective and time-efficient solution for biodiversity analysis in an environmental sample or clinical sample. In addition to biodiversity analysis, metagenomics sequencing focuses on biological functions and metabolic pathways.

Previous studies suggest that microbial markers in saliva may be useful in diagnosing and predicting some health conditions, such as oral cavity cancer. The role of bacteria in the development of HNSCC (head and neck squamous cell carcinoma) cancer has not been delineated, but the persistent presence of bacteria at tumor sites in the oropharynx and oral cavity raises attention to the issue.

Materials & Methods

Study patients

19 HNSCC patients

25 controls

Saliva and tumor samples

In situ hybridization

Quantitative PCR

HPV-16

HPV data analysis

V3-V5 amplicon libraries

16S amplicon sequencing

Data analysis

OTUs clustering

OTUs analysis

LEfSe algorithm

Longitudinal analyses

Results

1. Diversity analysis

As depicted in Figure 1, the beta diversity analysis discriminated HNSCC from control samples. PCA (Principal Component Analysis) reveals that the microbial communities in HNSCC patients are significantly different from those in control samples. NMDS revealed that the microbial communities in HPV- oropharyngeal samples are significantly different from those in HPV- oral cavity patients.

13 assigned phyla were identified, with 5 dominating across all of the samples: Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, and Fusobacteria. Genus-level profiles exhibited a dominance of Streptococcus and Prevotella in all samples. And some differences were observed between HNSCC samples and controls.

Figure 2. Taxonomic profiles at the phyla and genus levels.

3. The enrichment of Lactobacillus and Streptococcus in HNSCC

OTU analysis and LEfSe algorithm confirmed the enrichment of Lactobacillus and Streptococcus in HNSCC samples compared to controls.

• OTU network

The OTU network depicted in Figure 3 significantly discriminates the HNSCC samples from normal samples, and HPV negative from HPV positive samples. The total abundance of Streptococcus, Dialister, and Veillonella had a different dominance in tumor samples and control samples.

Figure 4. Differentially enriched microbiota OTUs in HNSCC when compared to control samples.

• LEfSe

The linear discriminant analysis for effect size (LEfSe) algorithm on genus level OTU tables confirmed the significant enrichment of Lactobacillus and Streptococcus in HNSCC samples compared to controls. Significant pre-treatment differences of saliva microbiome were observed between patients only treated with surgery to patients treated with chemo-radiation therapy and surgery (CRT/Surgery).

Figure 5. LDA Effect size (LEfSe) algorithm was used to determine taxa that best characterize each biological class.

• Longitudinal analyses of selected samples

Longitudinal analyses suggested that each patient had differentially abundant taxa between each time point. there was a decrease in Streptococcus and an increase in Lactobacillus as TNM stage progressed.

The results provide intriguing and preliminary data about the relationship between the salivary microbiome and head and neck cancer, HPV status, and the treatment. It suggests that the enriched presence of Lactobacillus, or the loss of Haemopilus, Neisseria, Gemellaceae or Aggregatibacter in saliva may be biomarkers for HNSCC cancer. The potential biomarkers for HPV+ HNSCC tumors may include Veillonella, Megasphaera and Anaerolineae.